The germline starts as the fertilized egg, or zygote. This cell divides into a cluster of physiologically identical blastomeres which form a hollow ball called the blastocyst. Several days later the dividing cells become differentially committed to form particular parts of the embryo. One group of cells, is set aside very early from the embryonic yolk sac to continue the germline, migrating to the gonads (testes and ovaries) and later form the gametes (sperm and eggs, which pass their genes to the next generation).
Somatic Cells make up the rest of the body. Changes to their genes do not pass to the next generation. Indeed, a central tenet of Mendelian genetics, the Weissman boundary, asserts that nothing that happens to the somatic cells or tissues of a mammal will have any effect whatsoever on the genetic information transmitted to its offspring. Many research laboratories are currently able to inject DNA successfully into fertilized eggs of frogs, mice and other mammals, but at this time all gene therapy in humans introduces DNA only into somatic cells. If germline gene therapy is ever perfected it will have certain advantages over somatic therapy because changes could be incorporated into every cell of the body, even those inaccessible to somatic techniques. Of course, with germline therapy geneticists would have to incorporate effective genetic control sequences along with the transgenes to ensure that they were expressed only in the intended cell types at the intended times. Germline engineering would have the distinct disadvantage, however, that its genetic modifications would be applied to the first cell of the embryo and hence (unlike somatic engineering) could not possibly be used to address problems later in adults.
Two stages of the germline are suitable for genetic engineering, the released egg, (before or after fertilization with sperm (when it is known as a Zygote) and cells at the developmental stage of blastomeres, which are the cells into which the egg divides during cleavage. Both the egg and blastomeres have attractive features for genetic engineering.
The egg is a very large cell, relatively easy to manipulate and inject with DNA. Remarkably, if DNA is injected into an egg, it will often stably integrate into one of the chromosomes, and therefore be incorporated into all subsequent cells of the body.
But, complications can arise. Sometimes a segment of DNA introduced into a fertilized egg will not become integrated into a chromosome until after the egg divides. Then only some of the cells of the embryo will contain a copy of that DNA. That resulting animal will be a mosaic of modified and unmodified cell types.
Also, several copies of the introduced DNA can become integrated into different chromosomes. In this case different genotypes will segregate during subsequent generations. In order to obtain a stable new strain, several generations of descendants must be examined and animals with correct genotype selected.
The blastomere stage is especially convenient for genetic engineering because lines of cells at this developmental stage can be grown and manipulated in the test tube. These cells, called embryonal stem (ES) cells, can be propagated indefinitely in cell culture. One or several ES cells can be injected into a blastocyst (obtained by culturing a fertilized egg for several division cycles in a Petri dish) and then implanted in a surrogate mother. Both types of cells in the hybrid blastocyst can contribute patches of cells to the final adult animal. This holds for both somatic and germline tissues.
The resulting animal is therefore a mosaic or chimera; an individual composed of a mixture of genetically different cells. The genotypes of the two types of cells remain distinct, they do not blend. Thus, individual sperm or eggs may be derived from either the ES cells or those of the fertilized egg. In the former case, the resulting progeny has the genetic constitution expected if the test tube of cultured ES cells had been one of its parents.
Embryonic stem cells have many advantages for genetic engineering. A culture of millions of identical cells can be treated with a DNA preparation. Once a suitable selection scheme is devised, a cell altered in a particular way can be plucked out from the huge excess of cells that integrated their DNA in inappropriate ways or at inappropiate locations on the chromosomes. It can be grown into a large homogeneous population whose genotype can in analyzed in detail. Concievably this culture can be subjected to a second round of genetic engineering, a third, fourth and so on. In addition, viable samples of the cultures can be preserved for long periods of time by freezing, and any number of mosaic embryos can be constructed from these engineered cell lines. Finally, the source of the egg used to obtain the blastocysts into which the ES cells are placed will not affect the final engineered animal. Eggs can come from "any old breeding stock".
Many of these same advantages also apply to technological extension that uses nuclear transfers. Instead of introducing the cultured cells into a blastocyst, the nucleus of one cultured cell is substituted for the nucleus of a fertilized egg. This technique has not been used extensively by genetic engineers but its spectacular recent use in producing clones of animals with identical genotype has sharply increased interest in it. One advantage is that an animal with the genetic constitution of a cultured cell can be obtained in one generation. Another is that cultures of a wider variety of cell types probably can be used. Some selection procedures may be easier to carry out on cultures of cells more differentiated than ES cells. The technique of nuclear transplantation pioneered to produce genetically identical clones may find its greatest importance in engineering changes in genomes.
Germline genetic engineering is still in its infancy. Already, however, it has been important for producing a variety of types of specially altered animals. Some examples are: